Efflux Might Participate in Decreased Susceptibility to Oxytetracycline in Contagious Agalactia-Causative Mycoplasma spp.

Contagious agalactia is associated with mastitis, keratoconjunctivitis, arthritis, pneumonia, and septicemia in small ruminants in countries with large dairy industries worldwide. The causative agents belong to four (sub)species of the Mycoplasma genus that have remained essentially susceptible to antimicrobials, including to the widely-used tetracycline family. However, some clinical isolates have been detected that show increased minimum inhibitory concentrations of tetracyclines, although they do not harbor the mutation in the 16SrRNA gene usually associated with resistance. The present work aimed to assess whether efflux pumps, infrequently described in mycoplasmas, could participate in the observed moderate loss of susceptibility. General efflux mechanisms were measured (i) using the fluorescence property of ethidium bromide when accumulated intracellularly and intercalated in the mycoplasma genomes, its active extrusion resulting in a temperature-dependent decrease in fluorescence and (ii) monitoring the growth inhibition of mycoplasmas by subinhibitory concentrations of tetracycline with or without reserpine, a known inhibitor of efflux in other bacteria. Both methods revealed non-specific efflux phenomena in most of the isolates tested, although their efficacy was difficult to quantify. This property could contribute to the acquisition of mutations conferring resistance by maintaining intracellular concentrations of tetracyclines at subinhibitory levels.

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A Survey of Mycoplasma Agalactiae in Small Ruminants with Contagious Agalactiae Syndrome in Iran

Bangladesh Journal of Veterinary Medicine ◽

10.3329/bjvm.v12i1.20466 ◽

2014 ◽

Vol 12 (1) ◽

pp. 67-72 ◽

Cited By ~ 1

Author(s):

M Khezri ◽

SA Pourbakhsh

Keyword(s):

Synovial Fluid ◽

Animal Health ◽

Economic Effect ◽

Small Ruminants ◽

Geographic Area ◽

Mycoplasma Agalactiae ◽

Contagious Agalactia ◽

Nasal Swabs ◽

Pcr Method ◽

Mycoplasma Spp

Contagious agalactia (CA) is one of the major animal health problems in small ruminants. It has economic effect and is caused by Mycoplasma agalactiae, the classic etiological agent in sheep and goats. The significance of the different Mycoplasma spp causing CA varies depending on the geographic area. This study conducted between 2012 and 2013 on 189 small ruminants with CA signs in the west of Iran, an area where CA is endemic. All samples (milk, synovial fluid, ear swabs, conjunctival swabs and nasal swabs) were examined by PCR method. Mycoplasma spp was detected in 76.2% and M. agalactiae isolated from 16% of positive samples. M. agalactiae were isolated from 7 conjunctival swabs, 15 milk samples and one synovial fluid sample. Results showed that M. agalactiae was found to be the non-main cause of CA in small ruminants in Iran.DOI: http://dx.doi.org/10.3329/bjvm.v12i1.20466 Bangl. J. Vet. Med. (2014). 12 (1): 67-72

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A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

The Open Microbiology Journal ◽

10.2174/1874285801307010072 ◽

2013 ◽

Vol 7 (1) ◽

pp. 72-82 ◽

Cited By ~ 39

Author(s):

Marta Martins ◽

Matthew P McCusker ◽

Miguel Viveiros ◽

Isabel Couto ◽

Séamus Fanning ◽

...

Keyword(s):

Ethidium Bromide ◽

Efflux Pump ◽

Efflux Pumps ◽

Outer Cell ◽

Gram Negative Bacteria ◽

Drug Resistant ◽

Topoisomerase Iv ◽

Simple Method ◽

Gram Negative ◽

Over Expression

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

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RND Efflux Systems Contribute to Resistance and Virulence of Aliarcobacter butzleri

Antibiotics ◽

10.3390/antibiotics10070823 ◽

2021 ◽

Vol 10 (7) ◽

pp. 823

Author(s):

Cristiana Mateus ◽

Ana Rita Nunes ◽

Mónica Oleastro ◽

Fernanda Domingues ◽

Susana Ferreira

Keyword(s):

Oxidative Stress ◽

Human Serum ◽

Ethidium Bromide ◽

Biofilm Formation ◽

Bacterial Resistance ◽

Efflux Pumps ◽

Bacterial Virulence ◽

Relative Fitness ◽

Mutant Strains

Aliarcobacter butzleri is an emergent enteropathogen that can be found in a range of environments. This bacterium presents a vast repertoire of efflux pumps, such as the ones belonging to the resistance nodulation cell division family, which may be associated with bacterial resistance, as well as virulence. Thus, this work aimed to evaluate the contribution of three RND efflux systems, AreABC, AreDEF and AreGHI, in the resistance and virulence of A. butzleri. Mutant strains were constructed by inactivation of the gene that encodes the inner membrane protein of these systems. The bacterial resistance profile of parental and mutant strains to several antimicrobials was assessed, as was the intracellular accumulation of the ethidium bromide dye. Regarding bacterial virulence, the role of these three efflux pumps on growth, strain fitness, motility, biofilm formation ability, survival in adverse conditions (oxidative stress and bile salts) and human serum and in vitro adhesion and invasion to Caco-2 cells was evaluated. We observed that the mutants from the three efflux pumps were more susceptible to several classes of antimicrobials than the parental strain and presented an increase in the accumulation of ethidium bromide, indicating a potential role of the efflux pumps in the extrusion of antimicrobials. The mutant strains had no bacterial growth defects; nonetheless, they presented a reduction in relative fitness. For the three mutants, an increase in the susceptibility to oxidative stress was observed, while only the mutant for AreGHI efflux pump showed a relevant role in bile stress survival. All the mutant strains showed an impairment in biofilm formation ability, were more susceptible to human serum and were less adherent to intestinal epithelial cells. Overall, the results support the contribution of the efflux pumps AreABC, AreDEF and AreGHI of A. butzleri to antimicrobial resistance, as well as to bacterial virulence.

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PK/PD Analysis of Marbofloxacin by Monte Carlo Simulation against Mycoplasma agalactiae in Plasma and Milk of Lactating Goats after IV, SC and SC-Long Acting Formulations Administration

Animals ◽

10.3390/ani11041104 ◽

2021 ◽

Vol 11 (4) ◽

pp. 1104

Author(s):

Emilio Fernández-Varón ◽

Edgar García-Romero ◽

Juan M. Serrano-Rodríguez ◽

Carlos M. Cárceles ◽

Ana García-Galán ◽

...

Keyword(s):

Monte Carlo Simulation ◽

Monte Carlo ◽

High Performance ◽

Small Ruminants ◽

Area Under The Curve ◽

Hplc Method ◽

Surrogate Markers ◽

Contagious Agalactia ◽

Lactating Goats ◽

Long Acting

Contagious agalactia is a mycoplasmosis affecting small ruminants that have become an important issue in many countries. However, PK/PD studies of antibiotics to treat this problem in lactating goats affected by Mycoplasma (M.) agalactiae, the main CA-causing mycoplasma are almost non-existent. The aims of this study were to evaluate the plasma and milk disposition of marbofloxacin in lactating goats after intravenous (IV), subcutaneous (SC) and subcutaneous poloxamer P407 formulations with and without carboxy-methylcellulose (SC-P407-CMC and SC-P407) administration. Marbofloxacin concentrations were analysed by the High Performance Liquid Chromatography (HPLC) method. Minimum inhibitory concentrations (MIC) of M. agalactiae field isolates from mastitic goat’s milk were used to calculate surrogate markers of efficacy. Terminal half-lives of marbofloxacin after IV, SC, SC-P407 and SC-P407-CMC administration were 7.12, 6.57, 13.92 and 12.19 h in plasma, and the half-lives of elimination of marbofloxacin in milk were 7.22, 7.16, 9.30 and 7.74 h after IV, SC, SC-P407 and SC-P407-CMC administration, respectively. Marbofloxacin penetration from the blood into the milk was extensive, with Area Under the Curve (AUCmilk/AUCplasma) ratios ranged 1.04–1.23, and maximum concentrations (Cmax-milk/Cmax-plasma) ratios ranged 0.72–1.20. The PK/PD surrogate markers of efficacy fAUC24/MIC and the Monte Carlo simulation show that marbofloxacin ratio (fAUC24/MIC > 125) using a 90% of target attainment rate (TAR) need a dose regimen between 8.4 mg/kg (SC) and 11.57 mg/kg (P407CMC) and should be adequate to treat contagious agalactia in lactating goats.

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Mycoplasma agalactiae: The Sole Cause of Classical Contagious Agalactia?

Animals ◽

10.3390/ani11061782 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1782

Author(s):

Sergio Migliore ◽

Roberto Puleio ◽

Robin A. J. Nicholas ◽

Guido R. Loria

Keyword(s):

Diagnostic Tests ◽

Clinical Signs ◽

Small Ruminants ◽

Molecular Methods ◽

The Other ◽

Animal Disease ◽

Mycoplasma Agalactiae ◽

Contagious Agalactia ◽

Mycoplasma Capricolum ◽

Mycoplasma Mycoides

Contagious agalactia (CA) is suspected when small ruminants show all or several of the following clinical signs: mastitis, arthritis, keratoconjunctivitis and occasionally abortion. It is confirmed following mycoplasma isolation or detection. The historical and major cause is Mycoplasma agalactiae which was first isolated from sheep in 1923. Over the last thirty years, three other mycoplasmas (Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens) have been added to the etiology of CA because they can occasionally cause clinically similar outcomes though nearly always in goats. However, only M. agalactiae is subject to animal disease regulations nationally and internationally. Consequently, it makes little sense to list mycoplasmas other than M. agalactiae as causes of the OIE-listed CA when they are not officially reported by the veterinary authorities and unlikely to be so in the future. Indeed, encouraging countries just to report M. agalactiae may bring about a better understanding of the importance of CA. In conclusion, we recommend that CA should only be diagnosed and confirmed when M. agalactiae is detected either by isolation or molecular methods, and that the other three mycoplasmas be removed from the OIE Manual of Diagnostic Tests and Vaccines in Terrestrial Animals and associated sources.

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An Investigation of Contagious Agalactia Disease of Sheep and Goats in Isparta and Afyonkarahisar in Turkey

Indian Journal of Animal Research ◽

10.18805/ijar.bf-1428 ◽

2021 ◽

Author(s):

Ülkü Karatekeli ◽

Beytullah Kenar

Keyword(s):

Polymerase Chain Reaction ◽

Growth Inhibition ◽

Colony Morphology ◽

Economic Losses ◽

Pcr Analysis ◽

Chain Reaction ◽

Biochemical Tests ◽

Contagious Agalactia ◽

Nasal Swabs ◽

Polymerase Chain

Background: Contagious agalactia causes significant economic losses. The aim of this study is to investigate the presence of contagious agalactia disease in cities of Isparta and Afyonkarahisar, Turkey. Methods: The study includes 45.500 animals in 220 ovine enterprises and samples were taken from those suspected of contagious agalactia disease. 202 animals in the 21 ovine enterprises comprised of 139 goats, 56 sheep, 3 kid goats, 2 goats, 2 lambs in total suspected of the disease were sampled. A total of 289 samples were collected, including 91 milk samples, 28 nasal swabs, 101 eye swabs, 8 joint fluids and 61 ear swabs. The isolates obtained after incubation were identified with polymerase chain reaction by using specific primers to assess film and spot formation, glucose fermentation, growth inhibition tests. Result: Three Mycoplasma spp. isolates obtained from 28 nasal swabs turned out to be negative for M. agalactiae after PCR analysis. Colony morphology, biochemical tests and growth inhibition tests revealed that one agent was M. arginine and the two factors were identified as M. ovipneumoniae with centerless colony morphology. The obtained results were confirmed with the polymerase chain reaction. None of the four factors causing contagious agalactia were isolated and identified.

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Functional Cloning and Characterization of a Multidrug Efflux Pump, MexHI-OpmD, from a Pseudomonas aeruginosa Mutant

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.9.2990-2992.2003 ◽

2003 ◽

Vol 47 (9) ◽

pp. 2990-2992 ◽

Cited By ~ 49

Author(s):

Hiroshi Sekiya ◽

Takehiko Mima ◽

Yuji Morita ◽

Teruo Kuroda ◽

Tohru Mizushima ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Ethidium Bromide ◽

Rhodamine 6G ◽

Efflux Pump ◽

Efflux Pumps ◽

Multidrug Efflux ◽

Chromosomal Dna ◽

Multidrug Efflux Pump ◽

Multidrug Efflux Pumps

ABSTRACT We isolated mutant YM644, which showed elevated resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G, from Pseudomonas aeruginosa YM64, a strain that lacks four major multidrug efflux pumps. The genes responsible for the resistance were mexHI-opmD. Elevated ethidium extrusion was observed with cells of YM644 and YM64 harboring a plasmid carrying the genes. Disruption of the genes in the chromosomal DNA of YM644 made the cells sensitive to the drugs.

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Ethidium Bromide MIC Screening for Enhanced Efflux Pump Gene Expression or Efflux Activity in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01058-10 ◽

2010 ◽

Vol 54 (12) ◽

pp. 5070-5073 ◽

Cited By ~ 49

Author(s):

Diixa Patel ◽

Christos Kosmidis ◽

Susan M. Seo ◽

Glenn W. Kaatz

Keyword(s):

Staphylococcus Aureus ◽

Ethidium Bromide ◽

Efflux Pump ◽

Large Strain ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Reverse Transcription Pcr ◽

Biocide Resistance ◽

Straightforward Method ◽

Short Period

ABSTRACT Multidrug resistance efflux pumps contribute to antimicrobial and biocide resistance in Staphylococcus aureus. The detection of strains capable of efflux is time-consuming and labor-intensive using currently available techniques. A simple and inexpensive method to identify such strains is needed. Ethidium bromide is a substrate for all but one of the characterized S. aureus multidrug-resistant (MDR) efflux pumps (NorC), leading us to examine the utility of simple broth microtiter MIC determinations using this compound in identifying efflux-proficient strains. Quantitative reverse transcription-PCR identified the increased expression of one or more MDR efflux pump genes in 151/309 clinical strains (49%). Ethidium bromide MIC testing was insensitive (48%) but specific (92%) in identifying strains with gene overexpression, but it was highly sensitive (95%) and specific (99%) in identifying strains capable of ethidium efflux. The increased expression of norA with or without other genes was most commonly associated with efflux, and in the majority of cases that efflux was inhibited by reserpine. Ethidium bromide MIC testing is a simple and straightforward method to identify effluxing strains and can provide accurate predictions of efflux prevalence in large strain sets in a short period of time.

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Rescue of streptomycin activity by piperine in Mycobacterium tuberculosis

Future Microbiology ◽

10.2217/fmb-2020-0124 ◽

2021 ◽

Author(s):

Leonora L Calsavara ◽

Laíse A Hegeto ◽

Eloisa G Sampiron ◽

Giovana F Costacurta ◽

Letícia S Murase ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Minimum Inhibitory Concentration ◽

Ethidium Bromide ◽

Inhibitory Concentration ◽

Efflux Pumps ◽

Clinical Isolates ◽

Relative Quantification

Aim: To evaluate the modulatory effect of piperine (PIP) on streptomycin (SM) activity in Mycobacterium tuberculosis ( Mtb). Materials & methods: SM and PIP minimum inhibitory concentration (MIC) and combinatory activity were determined in Mtb H37Rv and in susceptible and resistant clinical isolates. Ethidium bromide accumulation assay and relative quantification of efflux pumps genes ( rv1258c, rv1218c and rv2942), after SM and SM+PIP combination exposure, were also performed. Results: PIP concentration of 25 μg/ml (1/4× MIC) was able to inhibit efflux pumps activity, to modulate SM activity in Mtb, and conducted changes in the relative quantification of efflux pumps genes. Conclusion: SM+PIP combination was able to rescue the SM susceptible MIC values in SM resistant Mtb.

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Epizootological course of monieziosis of small cattle in the lowland regions of the Сhechen republic

International bulletin of Veterinary Medicine ◽

10.17238/issn2072-2419.2020.1.33 ◽

2020 ◽

Vol 1 ◽

pp. 33-36

Author(s):

H.H. Hakhbiev ◽

◽

N.I. Kosyaev ◽

I.Kh. Hakhbiev ◽

◽

...

Keyword(s):

Age Groups ◽

Small Ruminants ◽

Poor Quality ◽

Economic Damage ◽

North Caucasus ◽

Caucasus Region ◽

The North ◽

Condi Tions ◽

Causative Agents ◽

The Russian Federation

The invasion of small cattle by moniezio-sis in the Russian Federation occurs every-where and causes great economic damage to sheep farms, leading to the death of lambs and to decrease of meat and wool quality. In lambs infected with monieziosis, there is a lack in weight gain of 1.5 – 3 kg, as well as a decrease in the production of quality wool from a sheep to one kilogram. Damage from intestinal helminthozises, in particular ces-todosis, leads to death and forced slaughter, poor-quality wool and non-viable offspring. The dynamics of the epizootological process, patterns of the spread and course of the inva-sion in the North Caucasus region remain poorly studied, especially of the intestinal cestodes, namely monieziosis of small cat-tle. Taking into account the climate condi-tions of the Chechen Republic, and the lack of the knowledge of invasion, it is nec-essary to study natogeorgia and species com-position of causative agents of monieziosis of small ruminants, particularly sheep. The purpose of our research was to study the ecology and prevalence of monieziosis of sheep of different age groups in the plains of the Chechen Republic, to identify the species and age groups of monieziosis. In the flat areas of the Chechen Republic, infection with monieziosis determines the ecological balance of the parasitic structures of the ces-todes. The extensiveness and intensity of M. exports are compared with M. benedeni more. In lowland farms, lambs of small cat-tle have an EI of 12.9 – 16.7% with an EI of 2.2 ECZ / head.; young animals 9.1-11.1% and 2.1 ex/head.; in sheep, 4, 9 – 6.1% and 1.0-1.3 ex / head. EI M. benedeni lambs 7,3 – 8,1, AI-1,2 – 1,3 ex/goal.; young EI-5.6 -6.5% II-1ekz / goal. M. expansa was not found in mature sheep. In lowland areas, sheep monieziosis is observed with focal prevalence in all the farms studied by us during the year.

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